Defective mitophagy in human Niemann-Pick Type C1 neurons is due to abnormal autophagy activation

Abstract

Although traditionally regarded as a cellular adaptive process triggered by nutrient deprivation, autophagy in neurons appears to provide an important neuroprotective mechanism. Neurons in the brain are protected from starvation, and neuronal autophagy serves a critical role in the turnover of abnormal proteins and damaged organelles. As post-mitotic, highly polarized cells with active protein trafficking, neurons rely heavily on an efficient autophagic pathway. Using human embryonic stem cell-derived neurons engineered to mimic the cholesterol lysosomal storage disease Niemann Pick type C1 (NPC1), we have shown that excessive activation and impaired progression of the autophagic pathway conspire to cause abnormal mitochondrial clearance. Defective mitophagy is exceptionally severe in human NPC1 neurons, as compared with patient fibroblasts, and may explain the selective neuronal failure observed in NPC1 and related neurodegenerative disorders.

Due to its extreme efficiency, autophagy in neurons proceeds in the absence of easily detectable autophagic intermediates. For this reason, constitutive autophagy was initially thought not to be a prominent process in neurons. It is now clear that autophagy is essential in neurons and protects them from other types of stress, such as accumulation of abnormal proteins or dysfunctional organelles. Normal autophagy is essential to neuronal homeostasis, and autophagy dysfunction is a main contributor to various neurodegenerative disorders.

Because neurons are exceptionally reliant on autophagy, this is an attractive candidate to explain why neurons are particularly susceptible to defects that affect ubiquitous cellular pathways. Niemann Pick type C1 is an example of a disorder where sequestration of cholesterol in the lysosomal compartment is present in all cell types, but causes preferential neuronal failure. Prior studies implicated autophagy activation in the pathogenesis of NPC1 in mouse neurons and postmortem human brain slices. However, the dynamics of autophagy have never been analyzed in live human neurons, and identifying early events that lead to neuronal failure has been elusive. Analysis of live human neurons unlocks for the first time the possibility of developing therapies that can ameliorate the neurodegeneration seen in NPC1 and related disorders before significant neuronal death has occurred.

To exploit this possibility, we used human embryonic stem cell (hESC) technology and gene silencing to generate NPC1 knockdown neurons that replicate the pathology seen in NPC1. Using antibodies directed against the autophagy markers LC3-II and SQSTM1/p62, we found that human NPC1 neurons have strong spontaneous activation of autophagy, consistent with prior studies. Induction of autophagy would be expected to increase turnover of various cellular products, including mitochondria. However, using high resolution imaging of fluorescently labeled mitochondria, we found that downstream processing of autophagic intermediates is impaired in NPC1 neurons, which show a paradoxical accumulation of mitochondrial fragments. Mitochondrial fragmentation is an exceptionally severe phenotype in NPC1 neurons compared with patient fibroblasts, causing abnormal accumulation of mitochondrial proteins. We were able to rescue these abnormal phenotypes in NPC1 neurons by inhibiting autophagy and by mobilization of cholesterol from the lysosomal compartment. This latter observation suggests a mixed scenario where excessive activation and impaired progression of the autophagic pathway conspire to cause abnormal mitochondrial clearance.

A variety of reasons can explain why autophagy is excessively activated in human NPC1 neurons. Aberrant cholesterol distribution is likely to disrupt multiple cellular functions. As a result, autophagy may be required in order to clear abnormal proteins, organelles, or other cellular debris. However, an interesting question is whether NPC1 creates a unique scenario where lysosomal sequestration of cholesterol imposes a state of relative cholesterol deprivation. Although amino acid starvation is the best-known trigger for autophagy, there is evidence that lipid deprivation may independently stimulate autophagy activation. Therefore, autophagy activation may be neuroprotective in NPC1 by acting as an alternative route to release cholesterol from the lysosomal compartment. One can speculate that release of cholesterol from the autolysosome could occur by vesicular budding during the process of fusion between the lysosome and the autophagosome, or by contribution of lipid transporters supplied by the autophagosome. Although this scenario remains purely hypothetical, it is certain that neuronal survival would not be possible if the cholesterol supply were completely abolished. A matter of active research is to identify alternative pathways that mediate transfer of cholesterol between intracellular compartments, and autophagy is an attractive candidate.

Another important consideration when discussing therapeutic approaches for NPC1 and other disorders where autophagy defects have a proposed role is to understand the specific nature of these defects. Autophagy is a complex process that involves multiple steps, and identifying the basis for autophagy failure is crucial to design targeted therapies. In the case of NPC1, a lysosomal system heavily burdened with cholesterol is unable to mediate efficient turnover of mitochondria. Therefore, despite a proposed beneficial role of autophagy on intracellular redistribution of cholesterol, the added problem of mitochondrial fragmentation raises a complication to the use of autophagy induction as a therapeutic approach in NPC1. A possible solution to this problem is to identify strategies that specifically modulate mitophagy, while preserving the neuroprotective function of bulk autophagy.

Our data provides the first example, in human cells, of a process that causes preferential neuronal defects in a neurodegenerative disease. By identifying and reverting early events that lead to neuronal failure in NPC1, our approach highlights the efforts that are currently being undertaken to fulfill the promise of stem cell technology in the development of new treatments for various human degenerative diseases.

Ordonez MP, Roberts EA, Kidwell CU, Yuan SH, Plaisted WC, Goldstein LS. Disruption and therapeutic rescue of autophagy in a human neuronal model of Niemann Pick type C1. Hum Mol Genet. 2012;21:2651–62. doi: 10.1093/hmg/dds090.